The isolation of enzymes has been considerably improved in recent years by the technique of affinity chromatography. This method makes use of specific interactions between substances. For this purpose, a substance is covalently bonded as ligand onto an insoluble carrier (matrix). The ligand must be able to undergo an interaction, of the nature of a complex, with the substance which is to be isolated. The ligand retains only those substances which specifically react with it. Other substances are washed out. The retained substances can be eluted from the carrier material using a solution of unbonded ligand or, for example, using a salt gradient.
The success of an affinity chromatography depends on how well the interactions which naturally takes place between the substance which is to be isolated and the ligand are simulated. Thus, the choice of the matrix and the manner of immobilization of the ligand are important. The matrix ought to be hydrophilic and have good mechanical and chemical stability. Steric effects impeding the interaction can be favorably affected by spacers. Neither the matrix nor the spacer ought to give rise to non-specific adsorption.
For the isolation of a protein, it is of course most favorable to use a ligand which interacts only with one protein or with few proteins. The capacity of the adsorbent depends on the ligand loading of the matrix being sufficiently high. The chemical bonding ought to be as uniform and stable as possible, that is to say as difficult to hydrolyze as possible.
Affinity chromatography can be used for the isolation of proteins, for example from plasma, especially of antithrombin III. Immobilized sulfated polysaccharides have proved useful as affinity material for this purpose, especially carrier-bound heparin. However, it is known that, on immobilization of heparin and other sulfated polysaccharides, modifications of the ligand, with a reduction in biological activity, may occur.
The present invention had the object of preparing a material for affinity chromatography, by covalently bonding a sulfated polysaccharide to a carrier so that a material with high biological activity, ligand density and stability is obtained.